Technetium-99m labeled peptides for imaging

ABSTRACT

This invention relates to radiolabeled peptides and methods for producing such peptides. Specifically, the invention relates to peptides, methods and kits for making such peptides, and methods for using such peptides to image sites in a mammalian body labeled with technetium-99m (Tc-99m) via a radiolabel-binding moiety which forms a neutral complex with Tc-99m.

This is a divisional of application Ser. No. 07/871,282, filed Apr. 30,1992.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to radiodiagnostic reagents and peptides, andmethods for producing labeled radiodiagnostic agents. Specifically, theinvention relates to peptides, methods and kits for making suchpeptides, and methods for using such peptides to image sites in amammalian body labeled with technetium-99m (Tc-99m) via aradiolabel-binding moiety which forms a neutral complex with Tc-99m.

2. Description of the Prior Art

In the field of nuclear medicine, certain pathological conditions arelocalized, or their extent is assessed, by detecting the distribution ofsmall quantities of internally-administered radioactively labeled tracercompounds (called radiotracers or radiopharmaceuticals). Methods fordetecting these radiopharmaceuticals are known generally as imaging orradioimaging methods.

In radioimaging, the radiolabel is a gamma-radiation emittingradionuclide and the radiotracer is located using a gamma-radiationdetecting camera (this process is often referred to as gammascintigraphy). The imaged site is detectable because the radiotracer ischosen either to localize at a pathological site (termed positivecontrast) or, alternatively, the radiotracer is chosen specifically notto localize at such pathological sites (termed negative contrast).

A number of factors must be considered for optimal radioimaging inhumans. To maximize the efficiency of detection, a radionuclide thatemits gamma energy in the 100 to 200 keV range is preferred. To minimizethe absorbed radiation dose to the patient, the physical half-life ofthe radionuclide should be as short as the imaging procedure will allow.To allow for examinations to be performed on any day and at any time ofthe day, it is advantageous to have a source of the radionuclide alwaysavailable at the clinical site.

A variety of radionuclides are known to be useful for radioimaging,including ⁶⁷ Ga, ^(99m) Tc (Tc-99m), ¹¹¹ In, ¹²³ I, ¹⁶⁹ Yb or ¹⁸⁶ Re.Tc-99m is a preferred radionuclide because it emits gamma radiation at140 keV, it has a physical half-life of 6 hours, and it is readilyavailable on-site using a molybdenum-99/technetium-99m generator.

The sensitivity of imaging methods using radioactively-labeled peptidesis much higher than other radiopharmaceuticals known in the art, sincethe specific binding of the radioactive peptide concentrates theradioactive signal over the area of interest. Small synthetic peptidesthat bind specifically to targets of interest may be advantageously usedas the basis for radiotracers. This is because: 1. they may besynthesized chemically (as opposed to requiring their production in abiological system such as bacteria or mammalian cells, or theirisolation from a biologically-derived substance such as a fragment of aprotein); 2. they are small, hence non-target bound radiotracer israpidly eliminated from the body, thereby reducing background(non-target) radioactivity and allowing good definition of the target;and 3. small peptides may be readily manipulated chemically to optimizetheir affinity for a particular binding site.

Small readily synthesized labeled peptide molecules are preferred asroutinely-used radiopharmaceuticals. There is clearly a need for smallsynthetic labeled peptides that can be directly injected into a patientand will image pathological sites by localizing at such sites. Tc-99mlabeled small synthetic peptides offer clear advantages as radiotracersfor gamma scintigraphy, due to the properties of Tc-99m as aradionuclide for imaging and the utility of specific-binding smallsynthetic peptides as radiotracer molecules.

Radiolabeled peptides have been reported in the prior art.

Ege et al., U.S. Pat. No. 4,832,940 teach radiolabeled peptides forimaging localized T-lymphocytes.

Olexa et al., 1982, European Patent Application No. 823017009 disclose apharmaceutically acceptable radiolabeled peptide selected from FragmentE₁ isolated from cross-linked fibrin, Fragment E₂ isolated fromcross-linked fibrin, and peptides having an amino acid sequenceintermediate between Fragments E₁ and E₂.

Ranby et al., 1988, PCT/US88/02276 disclose a method for detectingfibrin deposits in an animal comprising covalently binding aradiolabeled compound to fibrin.

Hadley et al., 1988, PCT/US88/03318 disclose a method for detecting afibrin-platelet clot in vivo comprising the steps of (a) administeringto a patient a labeled attenuated thrombolytic protein, wherein thelabel is selectively attached to a portion of the thrombolytic proteinother than the fibrin binding domain; and (b) detecting the pattern ofdistribution of the labeled thrombolytic protein in the patient.

Lees et al., 1989, PCT/US89/01854 teach radiolabeled peptides forarterial imaging.

Sobel, 1989, PCT/US89/02656 discloses a method to locate the position ofone or more thrombi in an animal using radiolabeled, enzymaticallyinactive tissue plasminogen activator.

Stuttle, 1990, PCT/GB90/00933 discloses radioactively labeled peptidescontaining from 3 to 10 amino acids comprising the sequencearginine-glycine-aspartic acid (RGD), capable of binding to an RGDbinding site in vivo.

Maraganore et al., 1991, PCT/US90/04642 disclose a radiolabeled thrombusinhibitor comprising (a) a inhibitor moiety; (b) a linker moiety; and(c) and anion binding site moiety.

Rodwell et al., 1991, PCT/US91/03116 disclose conjugates of "molecularrecognition units" with "effector domains".

Tubis et al., 1968, Int. J. Appl. Rad. Isot. 19: 835-840 describelabeling a peptide with technetium-99m.

Sundrehagen, 1983, Int. J. Appl. Rad. Isot. 34: 1003 describes labelingpolypeptides with technetium-99m.

The use of chelating agents for radiolabeling polypeptides, and methodsfor labeling peptides and polypeptides with Tc-99m are known in theprior art and are disclosed in co-pending U.S. patent applications Ser.Nos. 07/653,012 and 07/807,062, which are hereby incorporated byreference.

Although optimal for radioimaging, the chemistry of Tc-99m has not beenas thoroughly studied as the chemistry of other elements and for thisreason methods of radiolabeling with technetium are not abundant. Tc-99mis normally obtained as Tc-99m pertechnetate (TcO₄ ⁻ ; technetium in the+7 oxidation state), usually from a molybdenum-99/technetium-99mgenerator. However, pertechnetate does not bind well to other compounds.Therefore, in order to radiolabel a peptide, Tc-99m pertechnetate mustbe converted to another form. Since technetium does not form a stableion in aqueous solution, it must be held in such solutions in the formof a coordination complex that has sufficient kinetic and thermodynamicstability to prevent decomposition and resulting conversion of Tc-99meither to insoluble technetium dioxide or back to pertechnetate.

Such coordination complexes of Tc-99m (in the +1 to +6 oxidation states)are known. However, many of these complexes are inappropriate forradiolabeling due to the molecular geometry of the coordination complex.For the purpose of radiolabeling, it is particularly advantageous forthe coordination complex to be formed as a chelate in which all of thedonor groups surrounding the technetium ion are provided by a singlechelating ligand. This allows the chelated Tc-99m to be covalently boundto a peptide through a single linker between the chelator and thepeptide.

These ligands are sometimes referred to as bifunctional chelating agentshaving a chelating portion and a linking portion. Such compounds areknown in the prior art.

Byrne et al., U.S. Pat. No. 4,434,151 describe homocysteinethiolactone-derived bifunctional chelating agents that can coupleradionuclides to terminal amino-containing compounds that are capable oflocalizing in an organ or tissue to be imaged.

Fritzberg, U.S. Pat. No. 4,444,690 describes a series oftechnetium-chelating agents based on 2,3-bis(mercaptoacetamido)propanoate.

Byrne et al., U.S. Pat. Nos. 4,571,430 describe novel homocysteinethiolactone bifunctional chelating agents for chelating radionuclidesthat can couple radionuclides to terminal amino-containing compoundsthat are capable of localizing in an organ or tissue to be imaged.

Byrne et al., U.S. Pat. Nos. 4,575,556 describe novel homocysteinethiolactone bifunctional chelating agents for chelating radionuclidesthat can couple radionuclides to terminal amino-containing compoundsthat are capable of localizing in an organ or tissue to be imaged.

Davison et al., U.S. Pat. No. 4,673,562 describe technetium chelatingcomplexes of bisamido-bisthio-ligands and salts thereof, used primarilyas renal function monitoring agents.

Nicolotti et al., U.S. Pat. No. 4,861,869 describe bifunctional couplingagents useful in forming conjugates with biological molecules such asantibodies.

Fritzberg et al., U.S. Pat. No. 4,965,392 describe various S-protectedmercaptoacetylglycylglycine-based chelators for labeling proteins.

Fritzberg et al., European Patent Application No. 86100360.6 describedithiol, diamino, or diamidocarboxylic acid or amine complexes usefulfor making technetium-labeled imaging agents.

Dean et al., 1989, PCT/US89/02634 describe bifunctional coupling agentsfor radiolabeling proteins and peptides.

Flanagan et al., European Patent Application No. 90306428.5 discloseTc-99m labeling of synthetic peptide fragments via a set of organicchelating molecules.

Albert et al., European Patent Application No. WO 91/01144 discloseradioimaging using radiolabeled peptides related to growth factors,hormones, interferons and cytokines and comprised of a specificrecognition peptide covalently linked to a radionuclide chelating group.

Dean, co-pending U.S. patent application Ser. No. 07/653,012 teachesreagents and methods for preparing peptides comprising a Tc-99mchelating group covalently linked to a specific binding peptide forradioimaging in vivo, and is hereby incorporated by reference.

(It is noted that all of these procedures would be expected to formanionic complexes of technetium in the +5 oxidation state.)

Baidoo & Lever, 1990, Bioconjugate Chem. 1: 132-137 describe a methodfor labeling biomolecules using a bisamine bisthiol group that gives acationic technetium complex.

It is possible to radiolabel a peptide by simply adding athiol-containing moiety such as cysteine or mercaptoacetic acid. Suchprocedures have been described in the prior art.

Schochat et al., U.S. Pat. No. 5,061,641 disclose direct radiolabelingof proteins comprised of at least one "pendent" sulfhydryl group.

Dean et al., co-pending U.S. patent application Ser. No. 07/807,062teach radiolabeling peptides via attached groups containing free thiols,and is incorporated herein by reference.

Goedemans et al., PCT Application No. WO 89/07456 describe radiolabelingproteins using cyclic thiol compounds, particularly 2-iminothiolane andderivatives.

Thornback et al., EPC Application No. 90402206.8 describe preparationand use of radiolabeled proteins or peptides using thiol-containingcompounds, particularly 2-iminothiolane.

Stuttle, PCT Application No. WO 90/15818 describes Tc-99m labeling ofRGD-containing oligopeptides.

Again it is noted that in all of these cases the expected Tc-99m labeledspecies would be an anionic complex.

The binding of certain peptides to their target entities is sensitive tocharge modification of the peptide. Thus, it is disadvantageous in somecases to radiolabel a peptide with Tc-99m via a chelator that will forma charged Tc-99m complex. It is advantageous in certain cases to use achelator that will form an electrically neutral or uncharged Tc-99mcomplex.

This invention provides chelators for Tc-99m which may be used toprepare Tc-99m labeled peptides in which the Tc-99m is held as a neutralchelate complex.

Some chelators said to form neutral Tc-99m complexes have been describedin the prior art.

Burns et al., 1985, European Patent Application 85104959.3 describebisamine bisthiol compounds for making small neutral Tc-99m brainimaging agents.

Kung et al., 1986, European Patent Application 86105920.2 describebisamine bisthiol compounds for making small neutral Tc-99m imagingagents.

Bergstein et al., 1988, European Patent Application 88102252.9 describebisamine bisthiol compounds for making small neutral Tc-99m brainimaging agents.

Bryson et al., 1988, Inorg. Chem. 27: 2154-2161 describe neutralcomplexes of technetium-99 which are unstable to excess ligand.

Misra et al., 1989, Tet. Let. 30: 1885-1888 describe bisamine bisthiolcompounds for radiolabeling purposes.

Bryson et al., 1990, Inorg. Chem. 29: 2948-2951 describe chelatorscontaining two amide groups, a thiol group and a substituted pyridinethat may form neutral Tc-99 complexes.

Taylor et al., 1990, J. Nucl. Med. 31: 885 (Abst) describe a neutralTc-99m complex for brain imaging.

SUMMARY OF THE INVENTION

The present invention provides scintigraphic imaging agents that areradioactively-labeled peptides. The radiolabeled peptides of theinvention are comprised of peptides that specifically bind to a targetin vivo and are covalently linked to a radiolabel-binding moiety whereinthe moiety binds a radioisotope. It is a particular advantage in thepresent invention that the complex of the radiolabel-binding moiety andthe radiolabel is electrically neutral, thereby avoiding interference ofthe covalently linked radiolabeled complex with the specific bindingproperties of the specific binding peptide.

In a first aspect of the present invention, radiolabeled peptides areprovided capable of imaging sites within a mammalian body. The peptidesare comprised of a specific binding peptide having an amino acidsequence and a radiolabel-binding moiety covalently linked to thepeptide. Further, the complex of the radiolabel-binding moiety and theradiolabel is electrically neutral. In a preferred embodiment, thepeptide is covalently linked to the radiolabel-binding moiety via anamino acid, most preferably glycine. In another preferred embodiment,the radiolabel is technetium-99m.

In a second embodiment, the invention provides a radiolabeled peptidefor imaging sites within a mammalian body, comprising a specific bindingpeptide and a radiolabel-binding moiety of formula: ##STR1## (forpurposes of this invention, radiolabel-binding moieties having thisstructure will be referred to as picolinic acid (Pic)-based moieties) or##STR2## (for purposes of this invention, radiolabel-binding moietieshaving this structure will be referred to as picolylamine (Pica)-basedmoieties)

wherein X is H or a protecting group; (amino acid) is any amino acid;the radiolabel-binding moiety is covalently linked to the peptide andthe complex of the radiolabel-binding moiety and the radiolabel iselectrically neutral. In a preferred embodiment, the amino acid isglycine and X is an acetamidomethyl protecting group. In additionalpreferred embodiments, the peptide is covalently linked to theradiolabel-binding moiety via an amino acid, most preferably glycine,and the radiolabel is technetium-99m.

In yet another embodiment of the invention, a radiolabeled peptide isprovided for imaging sites within a mammalian body, comprising aspecific binding peptide and a bisamino bisthiol radiolabel-bindingmoiety covalently linked to the peptide. The bisamino bisthiolradiolabel-binding moiety in this embodiment of the invention has aformula selected from the group consisting of: ##STR3## wherein each Rcan be independently H, CH₃ or C₂ H₅ ; each (pgp)s can be independentlya thiol protecting group or H; m, n and p are independently 2 or 3; A islinear or cyclic lower alkyl, aryl, heterocyclyl, combinations orsubstituted derivatives thereof; and X is peptide; ##STR4## wherein eachR is independently H, CH₃ or C₂ H₅ ; m, n and p are independently 2 or3; A is linear or cyclic lower alkyl, aryl, heterocyclyl, combinationsor substituted derivatives thereof; V is H or CO-peptide; R' is H orpeptide; provided that when V is H, R' is peptide and when R' is H, V isCO-peptide. (For purposes of this invention, radiolabel-binding moietieshaving these structures will be referred to as "BAT" moieties). In apreferred embodiment, the peptide is covalently linked to theradiolabel-binding moiety via an amino acid, most preferably glycine,and the radiolabel is technetium-99m.

Specific-binding peptides provided by the invention include but are notlimited to peptides having the following sequences:

formyl-MLF

(VGVAPG)₃ amide SEQ ID NO:1

(VPGVG)₄ amide SEQ ID NO:2

RALVDTLKFVTQAEGAKamide SEQ ID NO:3

RALVDTEFKVKQEAGAKamide SEQ ID NO:4

PLARILPDFRLPEIAIPamide SEQ ID NO:5

GQQHHLGGAKAGDV SEQ ID NO:6

PLYKKIIKKLLES SEQ ID NO:7

LRALVDTLKamide SEQ ID NO:8

GGGLRALVDTLKamide SEQ ID NO:9

GGGLRALVDTLKFVTQAEGAKamide SEQ ID NO:11

GGGRALVDTLKALVDTLamide SEQ ID NO:11

GHRPLDKKREEAPSLRPAPPPISGGGYR SEQ ID NO:12

PSPSPIHPAHHDRRQamide SEQ ID NO:13

GGGF_(D).Cpa.YW_(D) KTFTamide SEQ ID NO:14 ##STR5##SYNRGDSTC(S-maleimidoCH₂ CH₂ --)!₃ N GGGLRALVDTLKamide SEQ ID NO:16

GCGGGLRALVDTLKamide SEQ ID NO:17

GCYRALVDTLKFVTQAEGAKamide SEQ ID NO:18

GC(VGVAPG)₃ amide SEQ ID NO:19

The invention also comprises complexes of the peptides of the inventionwith Tc-99m and methods for radiolabeling the peptides of the inventionwith Tc-99m. Radiolabeled complexes provided by the invention are formedby reacting the peptides of the invention with Tc-99m in the presence ofa reducing agent. Preferred reducing agents include but are not limitedto dithionite ion, stannous ion, and ferrous ion. Complexes of theinvention are also formed by labeling the peptides of the invention withTc-99m by ligand exchange of a prereduced Tc-99m complex as providedherein.

The invention also provides kits for preparing the peptides of theinvention radiolabeled with Tc-99m. Kits for labeling the peptide of theinvention with Tc-99m are comprised of a sealed vial containing apredetermined quantity of a peptide of the invention and a sufficientamount of reducing agent to label the peptide with Tc-99m.

This invention provides methods for preparing peptides of the inventionby chemical synthesis in vitro. In a preferred embodiment, peptides aresynthesized by solid phase peptide synthesis.

This invention provides methods for using Tc-99m labeled peptides forimaging a site within a mammalian body by obtaining in vivo gammascintigraphic images. These methods comprise administering an effectivediagnostic amount of a Tc-99m radiolabeled peptide of the invention anddetecting the gamma radiation emitted by the Tc-99m localized at thesite within the mammalian body.

Compositions of matter comprising radiolabel-binding moieties that forman electrically neutral complex with a radioisotope are also provided bythe invention. In a preferred embodiment, the radioisotope is Tc-99m.Additional preferred embodiments include bisamine, bisthiol derivativesand picolinic acid and picolylamine derivatives described herein.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides Tc-99m labeled peptides for imagingtarget sites within a mammalian body comprising an amino acid sequencecovalently linked to a radiolabel-binding moiety wherein theradiolabel-binding moiety binds a radioisotope and forms an electricallyneutral complex.

Labeling with Tc-99m is an advantage of the present invention becausethe nuclear and radioactive properties of this isotope make it an idealscintigraphic imaging agent. This isotope has a single photon energy of140 keV and a radioactive half-life of about 6 hours, and is readilyavailable from a ⁹⁹ Mo-^(99m) Tc generator. Other radionuclides known inthe prior art have effective half-lives which are much longer (forexample, ¹¹¹ In, which has a half-life of 67.4 h) or are toxic (forexample, ¹²⁵ I).

In the radiolabel binding moieties and peptides covalently linked tosuch moieties that contain a thiol covalently linked to a thiolprotecting groups ((pgp)^(S)) provided by the invention, thethiol-protecting groups may be the same or different and may be but arenot limited to:

--CH₂ -aryl (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH-(aryl)₂, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--C--(aryl)₃, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH₂ -(4-methoxyphenyl);

--CH--(4-pyridyl)(phenyl)₂ ;

--C(CH₃)₃

-9-phenylfluorenyl;

--CH₂ NHCOR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --NHCOOR (R is unsubstituted or substituted alkyl or aryl);

--CONHR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ -S--CH₂ -phenyl

Preferred protecting groups have the formula --CH₂ --NHCOR wherein R isa lower alkyl having 1 and 8 carbon atoms, phenyl or phenyl-substitutedwith lower alkyl, hydroxyl, lower alkoxy, carboxy, or loweralkoxycarbonyl. The most preferred protecting group is anacetamidomethyl group.

Peptides of the present invention can be chemically synthesized invitro. Peptides of the present invention can generally advantageously beprepared on a peptide synthesizer. The peptides of this invention can besynthesized wherein the radiolabel-binding moiety is covalently linkedto the peptide during chemical synthesis in vitro, using techniques wellknown to those with skill in the art. Such peptides covalently-linked tothe radiolabel-binding moiety during synthesis are advantageous becausespecific sites of covalent linkage can be determined.

Radiolabel binding moieties of the invention may be introduced into thetarget specific peptide during peptide synthesis. For embodiments (e.g.,Pic-Gly-Cys(protecting group)-) comprising picolinic acid (Pic-), theradiolabel-binding moiety can be synthesized as the last (i.e.,amino-terminal) residue in the synthesis. In addition, the picolinicacid-containing radiolabel-binding moiety may be covalently linked tothe ε-amino group of lysine to give, for example,αN(Fmoc)-Lys-εN(Pic-Gly-Cys(protecting group)), which may beincorporated at any position in the peptide chain. This sequence isparticularly advantageous as it affords an easy mode of incorporationinto the target binding peptide.

Similarly, the picolylamine (Pica)-containing radiolabel-binding moiety(-Cys(protecting group)-Gly-Pica) can be prepared during peptidesynthesis by including the the sequence (- Cys(protecting group)-Gly-)at the carboxyl terminus of the peptide chain. Following cleavage of thepeptide from the resin the carboxyl terminus of the peptide is activatedand coupled to picolylamine. This synthetic route requires that reactiveside-chain functionalities remain masked (protected) and do not reactduring the conjugation of the picolylamine.

Examples of small synthetic peptides containing the Pic-Gly-Cys- and-Cys-Gly-Pica chelators are provided in the Examples hereinbelow. Thisinvention provides for the incorporation of these chelators intovirtually any peptide, resulting in a radiolabeled peptide having Tc-99mheld as neutral complex.

This invention also provides specific-binding small synthetic peptideswhich incorporate bisamine bisthiol (BAT) chelators which may be labeledwith Tc-99m, resulting in a radiolabeled peptide having Tc-99m held asneutral complex. Examples of small synthetic peptides containing theseBAT chelators as radiolabel-binding moeity are provided in the Exampleshereinbelow.

In forming a complex of radioactive technetium with the peptides of thisinvention, the technetium complex, preferably a salt of Tc-99mpertechnetate, is reacted with the peptides of this invention in thepresence of a reducing agent. Preferred reducing agents are dithionite,stannous and ferrous ions; the most preferred reducing agent is stannouschloride. In an additional preferred embodiment, the reducing agent is asolid-phase reducing agent. Complexes and means for preparing suchcomplexes are conveniently provided in a kit form comprising a sealedvial containing a predetermined quantity of a peptide of the inventionto be labeled and a sufficient amount of reducing agent to label thepeptide with Tc-99m. Alternatively, the complex may be formed byreacting a peptide of this invention with a pre-formed labile complex oftechnetium and another compound known as a transfer ligand. This processis known as ligand exchange and is well known to those skilled in theart. The labile complex may be formed using such transfer ligands astartrate, citrate, gluconate or mannitol, for example. Among the Tc-99mpertechnetate salts useful with the present invention are included thealkali metal salts such as the sodium salt, or ammonium salts or loweralkyl ammonium salts.

In a preferred embodiment of the invention, a kit for preparingtechnetium-labeled peptides is provided. The peptides of the inventioncan be chemically synthesized using methods and means well-known tothose with skill in the art and described hereinbelow. Peptides thusprepared are comprised of between 3 and 100 amino acid residues, and arecovalently linked to a radiolabel-binding moiety wherein theradiolabel-binding moiety binds a radioisotope. An appropriate amount ofthe peptide is introduced into a vial containing a reducing agent, suchas stannous chloride or a solid-phase reducing agent, in an amountsufficient to label the peptide with Tc-99m. An appropriate amount of atransfer ligand as described (such as tartrate, citrate, gluconate ormannitol, for example) can also be included. Technetium-labeled peptidesaccording to the present invention can be prepared by the addition of anappropriate amount of Tc-99m or Tc-99m complex into the vials andreaction under conditions described in Example 3 hereinbelow.

Radioactively labeled peptides provided by the present invention areprovided having a suitable amount of radioactivity. In forming Tc-99mradioactive complexes, it is generally preferred to form radioactivecomplexes in solutions containing radioactivity at concentrations offrom about 0.01 millicurie (mCi) to 100 mCi per mL.

Technetium-labeled peptides provided by the present invention can beused for visualizing sites in a mammalian body. In accordance with thisinvention, the technetium-labeled peptides or neutral complexes thereofare administered in a single unit injectable dose. Any of the commoncarriers known to those with skill in the art, such as sterile salinesolution or plasma, can be utilized after radiolabeling for preparingthe injectable solution to diagnostically image various organs, tumorsand the like in accordance with this invention. Generally, the unit doseto be administered has a radioactivity of about 0.01 mCi to about 100mCi, preferably 1 mCi to 20 mCi. The solution to be injected at unitdosage is from about 0.01 mL to about 10 mL. After intravenousadministration, imaging of the organ or tumor in vivo can take place ina matter of a few minutes. However, imaging can take place, if desired,in hours or even longer, after the radiolabeled peptide is injected intoa patient. In most instances, a sufficient amount of the administereddose will accumulate in the area to be imaged within about 0.1 of anhour to permit the taking of scintiphotos. Any conventional method ofscintigraphic imaging for diagnostic purposes can be utilized inaccordance with this invention.

The technetium-labeled peptides and complexes provided by the inventionmay be administered intravenously in any conventional medium forintravenous injection such as an aqueous saline medium, or in bloodplasma medium. Such medium may also contain conventional pharmaceuticaladjunct materials such as, for example, pharmaceutically acceptablesalts to adjust the osmotic pressure, buffers, preservatives and thelike. Among the preferred media are normal saline and plasma.

The methods for making and labeling these compounds are more fullyillustrated in the following Examples. These Examples illustrate certainaspects of the above-described method and advantageous results. TheseExamples are shown by way of illustration and not by way of limitation.

EXAMPLE 1 Synthesis of BAT Chelators

A. Synthesis ofN-Boc-N'-(5-carboxypentyl)-N,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine

a. 2-methyl-2-(triphenylmethylthio)propanal

Triphenylmethylmercaptan (362.94 g, 1.31 mol, 100 mol %) dissolved inanhydrous THF (2 L) was cooled in an ice bath under argon. Sodiumhydride (60% in oil; 54.39 g, 1.35 mol, 104 mol %) was added in portionsover 20 min. 2-bromo-2-methylpropanal (206.06 g, 1.36 mol, 104 mol %;see Stevens & Gillis, 1957, J. Amer. Chem. Soc. 79: 3448-51) was thenadded slowly over 20 min. The reaction mixture was allowed to warm toroom temperature and stirred for 12 hours. The reaction was quenchedwith water (1 L) and extracted with diethyl ether (3×1 L). The etherextracts were combined, washed with saturated NaCl solution (500 mL),dried over Na₂ SO₄ and filtered. The solvent was removed under reducedpressure to afford a thick orange oil. The crude oil was dissolved intoluene (200 mL) and diluted to 2 L with hot hexanes. The mixture wasfiltered through a sintered glass funnel and cooled at -5° C. for 12hours. The white crystalline solid which formed was removed byfiltration to afford 266.36 g (59% yield) of the title compound. Themelting point of the resulting compound was determined to be 83°-85° C.Nuclear magnetic resonance characterization experiments yielded thefollowing molecular signature:

¹ H NMR(300 MHz, CDCl₃): δ 1.24(s, 6H, 2CH₃), 7.2-7.35 (m, 9H),7.59-7.62 (m, 6H), 8.69 (s, H, --COH)

¹³ C NMR (75 MHZ, CDCl₃): δ 22.86, 55.66, 67.48, 126.85, 127.75, 129.72,144.79, 197.31.

b. N,N'-Bis(2-methyl-2-(triphenylmethvlthio)propyl)ethylenediamine.

Ethylenediamine (1.3 mL, 0.0194 mol, 100 mol %) was added to2-methyl-2-(triphenylmethylthio)propanal (13.86 g, 0.0401 mol, 206 mol%) dissolved in methanol (40 mL) and anhydrous THF (40 mL) under argon,and the pH was adjusted to pH 6 by dropwise addition of acetic acid. Thesolution was stirred for 20 min at 20° C. Sodium cyanoborohydride (1.22g, 0.0194 mol, 100 mol %) was added and the reaction was stirred at roomtemperature for 3 hours. Additional sodium cyanoborohydride (1.08 g) wasadded and the reaction was stirred at 20° C. for 17 hours. A finalportion of sodium cyanoborohydride (1.02 g) was added and the reactionheated at reflux under argon for 6 hours. The reaction was quenched with0.5M HCl (100 mL) and extracted with ethyl acetate (2×100 mL). Theorganic extracts were combined, sequentially washed with 2M NaOH (60mL), saturated NaCl solution (60 mL), dried (Na₂ SO₄), and filtered. Thesolvent was removed under reduced pressure to give 16.67 g of crudeproduct which was crystallized from toluene/hexanes to afford 10.20 g(73% yield) of white crystals of the title compound. The melting pointof the resulting compound was determined to be 83°-86° C. FABMS analysisyielded an m/z of 721 (MH+). Nuclear magnetic resonance characterizationexperiments yielded the following molecular signature:

¹ H NMR (300 MHz, CDCl₃): δ 1.12 (s, 12H, 4 CH₃), 1.64 (s, 4H, N--CHC(Me)₂ S), 2.52 (s, 4H, N--CH₂ --CH₂ --N), 5.31 (S, 2H, 2-NH), 7.12-7.30(m, 18H, Ar), 7.62-7.65 (m, 12H, Ar).

c.N-(5-carboethoxypentyl)-N,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine

K₂ CO₃ (1.92 g, 13.9 mmol, 100 mol %) was added toN,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine (10.03 g,13.9 mmol) in CH₃ CN (60 mL), followed by ethyl 5-bromovalerate (3.30mL, 20.8 mmol, 150 mol %). The reaction was heated at reflux under argonovernight. The solution was then concentrated to a paste and partitionedbetween 0.25M KOH (100 mL) and ethyl acetate (100 mL). The aqueous layerwas extracted with ethyl acetate (1×50 mL) and the combined ethylacetate layers were washed with 50 mL water and NaCl solution (2×50 mL),dried with Na₂ SO₄ and concentrated to an orange oil. Purification byflash chromatography (300 g flash silica, 100% CHCl₃ to 5% MeOH/CHCl₃)gave pure title compound (7.75 g, 66% yield). FABMS analysis yielded an(MH+) of 849 (compared with a calculated molecular weight of 849.24 forthe compound C₅₅ H₆₄ N₂ O₂ S₂)

d.N-Boc-N'-(5-carboxypentyl)-N,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine

1M KOH (25 mL, 25.0 mmol, 274 mol %) was added toN-(5-carboethoxypentyl)-N,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine(7.75g, 9.13 mmol) in dioxane (200 mL), followed by water (250 mL). Dioxanewas then added dropwise with stirring until a homogeneous solution wasobtained. The reaction was heated at a slow reflux overnight. Most ofthe dioxane was removed by rotary evaporation and the pH of solution wasadjusted to ˜7-8 with 1 M KH₂ PO₄ and saturated NaHCO₃. The solution wasthen extracted with ethyl acetate (3×75 mL) and the combined organiclayers were washed with NaCl solution (50 mL), dried with Na₂ SO₄ andconcentrated to a foam/solid (6.35 g, 85% yield).

To the crude product from the above reaction was added (BOC)₂ O (3.35 g,15.4 mmol, 200 mol %), CH₃ CN (50 mL) and methylene chloride (50 mL),followed by triethylamine (1.0 mL, 7.2 mmol, 93 mol %). The reaction wasstirred at room temperature under argon overnight. The reaction solutionwas then concentrated and partitioned between water (100 mL) and ethylacetate (50 mL). The aqueous layer was extracted with ethyl acetate(1×50 mL) and the combined ethyl acetate layers were washed with 5%citric acid and NaCl solution (50 mL each), then dried (Na2SO₄) andconcentrated to an orange oil.

Purification by flash chromatography (200 g flash silica, 100% CDCl₃ to5% methanol/chloroform) gave pure title compound (2.58 g, 36% yield).FABMS analysis gave an (MH+) of 921 (compared with the calculated valueof 921.31 for the compound C₅₈ H₆₈ N₂ O₄ S₂).

B. Synthesis ofN-Boc-N'-(5-carboxypentyl)-N,N'-bis-(2-(4-methoxybenzylthio)-2-methylpropyl)ethylenediamine

a. N,N'-bis-(2-(4-methoxybenzylthio)-2-methylpropyl)-ethylenediamine

A solution of N,N'-bis(2-mercapto-2-methylpropyl)ethylene diamine (11.23g, 47.5 mmol; see, DiZio et al., 1991, Bioconjugate Chem 2: 353 andCorbin et al., 1976, J. Org. Chem. 41: 489) in methanol (500 mL) wascooled in ice/water bath and then saturated with gaseous ammonia over 45min. To this was added 4-methoxybenzyl chloride (17.0 mL, 125 mmol, 264mol %). The reaction was allowed to warm to room temperature overnightwith stirring under argon. The solution was concentrated to a paste andthen partitioned between diethyl ether (150 mL) and 0.5M KOH (200 mL).The aqueous layer was further extracted with diethyl ether (2×50 mL).The combined organic layers were washed with NaCl solution andconcentrated to a clear colorless oil. The oil dissolved in diethylether (200 mL) and then acidified with 4.0M HCl in dioxane until nofurther precipitation was seen. The white precipitate was collected byfiltration and washed with diethyl ether. The white solid wasrecrystallized from hot water at a pH of -2. The product was collectedby filtration to afford 29.94 g as a mix of mono- and di- HCl salts. TheHCl salts were partitioned between 1M KOH (100 mL) and ethyl acetate(100 mL). The aqueous was extracted with ethyl acetate (2×30 mL) and thecombined organic layers were washed with NaCl solution, dried with Na₂SO₄ and concentrated to give pure product as the free base as a lightyellow oil (18.53 g, 82% yield). Nuclear magnetic resonancecharacterization experiments yielded the following molecular signature:

¹ H NMR (300 MHz, CDCl): δ 7.25 (d, 4H, J=9), 6.83 (d, 4H, J=9), 3.78(s, 6H), (s, 4H), 2.63 (s, 4H), 2.56 (s, 4H), 1.34 (s, 12H).

b.N-(5-carboethoxypentyl)-N,N'-bis-(2-(4-methoxybenzylthio)-2-methylpropyl)ethylenediamine

To N,N'-bis- 2-(4-methoxybenzylthio)-2-methylpropyl!-ethylenediamine(4.13 g, 8.66 mmol) in CH₃ CN (50 mL) was added K₂ CO₃ (1.21 g, 8.75mmol, 101 mol %) followed by ethyl 5-bromovalerate (2.80 mL, 17.7 mmol,204 mol %). The reaction was stirred at reflux overnight and was thenconcentrated to a paste in vacuo. The residue was partitioned betweenethyl acetate (100 mL) and 0.5M KOH (100 mL). The aqueous layer wasextracted with ethyl acetate (1×50 mL) and the combined organic layerswere washed with NaCl solution (50 mL), dried with Na₂ SO₄ andconcentrated to a yellow oil (-6 g). Purification by normal-phasepreparative HPLC (100% CHCl₃ to 5% methanol/chloroform over 25 min.)afforded pure title compound (1.759 g, 34% yield). FABMS analysis gavean (MH+) of 605 (compared with the value of 604.90 calculated for C₃₃H₅₂ N₂ O₄ S₂). Nuclear magnetic resonance characterization experimentsyielded the following molecular signature:

¹ H NMR (300 mHz, CDCl₃): δ 7.25 (d, 4H, J=8.5), 6.83 (d, 4H, J=8.5),4.13 (q, J=7), 3.793 (s, 3H), 3.789 (s. 3H), 3.74 (s, 2H), 3.67 (s, 2H),2.6 (m, J=7), 1.6 (m, 2H), 1.5 (m 2H), 1.34 (s 12H), 1.28 (t, 3H, J=7).

c.N-Boc-N'-(5-carboxypentyl)-N,N'-bis-(2-(4-methoxybenzylthio)-2-methylpropyl)ethylenediamine

ToN-(5-carboethoxypentyl)-N,N'-bis-(2-(4-methoxybenzylthio)-2-methylpropyl)ethylenediamine(586 mg, 0.969 mmol) in THF (40 mL) was added water (30 mL) and 1M KOH(2.5 mL, 2.5 mmol, 260 mol %). The homogeneous solution was heated to aslow reflux overnight. The solution was then cooled to room temperatureand the THF was removed under rotary evaporation. The residue wasdiluted to 50 mL with H₂ O and the pH was adjusted to ˜2-3 with 1M HCl.The solution was extracted with ethyl acetate (3×30 mL) and the combinedorganic layers were washed with NaCl solution (50 mL), dried with Na₂SO₄ and concentrated to give crude acid (422 mg, 75% yield).

To the crude product from the above reaction was added CH₃ CN (40 mL)and (BOC)₂ O (240 mg, 1.10 mmol, 150 mol %) followed by triethylamine(0.200 mL, 1.43 mmol, 196 mol %). The homogenous solution stirred atroom temperature overnight under argon. The solution was thenconcentrated to a paste and partitioned between ethyl acetate (25 mL)and 1M KH2PO₄ (25 mL). The organic layer was washed with 5% citric acid(2×25 mL) and NaCl solution (25 mL), dried with Na₂ SO₄ and concentratedto a yellow oil. Purification by flash chromatography (50 mL flashsilica gel, 100% chloroform to 15% methanol/chloroform) gave pure titlecompound (344 mg, 70% yield). FABMS analysis gave an (MH+) of 677(compared to the value of 676.97 calculated for the compound C₃₆ H₅₆ N₂O₆ S₂). Nuclear magnetic resonance characterization experiments yieldedthe following molecular signature:

¹ H NMR (300 Mz, CDCl₃): δ 7.20 (d, 4H, J=7), 6.79 (d, 4H, J=7), 3.75(S, 3H), 3.74 (S, 3H), 3.68 (M, 4H), 3.35 (M, 4H), 2.65 (M, 2H), 2.53(M, 4H), 2.31 (M, 2H), 1.59 (M, 2H), 1.43 (S, 11H), 1.30 (S, 6H), 1.26(S, 6H)

EXAMPLE 2 Solid Phase Peptide Synthesis

Solid phase peptide synthesis (SPPS) was carried out on a 0.25 millimole(mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizerand using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection,coupling with dicyclohexylcarbodiimide/hydroxybenzotriazole or2-(1H-benzo-triazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate/hydroxybenzotriazole (HBTU/HOBT), and usingp-hydroxymethylphenoxymethyl-polystyrene (HMP) resin forcarboxyl-terminus acids or Rink amide resin for carboxyl-terminusamides. Resin-bound products were routinely cleaved using a solutioncomprised of trifluoroacetic acid, water, thioanisole, ethanedithiol,and triethylsilane, prepared in ratios of 100:5:5:2.5:2 for 1.5-3 h atroom temperature.

Where appropriate αN-formyl groups were introduced by treating thecleaved, deprotected peptide with excess acetic anhydride in 98% formicacid and stirring for about 18 hours followed by HPLC purification.Where appropriate N-terminal acetyl groups were introduced by treatingthe free N-terminal amino peptide bound to the resin with 20% v/v aceticanhydride in NMP (N-methylpyrrolidinone) for 30 min. Where appropriateBAT ligands were introduced either by using the appropriate BAT acid asthe last residue to be coupled during SPPS or by treating the N-terminusfree amino peptide bound to the resin with the BATacid/diisopropylcarbodiimide/N-hydroxysuccinimide in NMP. Whereappropriate, Cys-Cys disulfide bond cyclizations were performed bytreating the precursor cysteine-free thiol peptides at 0.1 mg/mL in pH 7buffer with aliquots of 0.006M K₃ Fe(CN)₆ until a stable yellow colorpersisted. The excess oxidant was reduced with excess cysteine, themixture was lyophilized and then purified by HPLC.

Crude peptides were purified by preparative high pressure liquidchromatography (HPLC) using a Waters Delta Pak C18 column and gradientelution using 0.1% trifluoroacetic acid (TFA) in water modified withacetonitrile. Acetonitrile was evaporated from the eluted fractionswhich were then lyophilized. The identity of each product was confirmedby fast atom bombardment mass spectroscopy (FABMS).

EXAMPLE 3 A General Method for Radiolabeling with Tc-99m

0.1 mg of a peptide prepared as in Example 2 was dissolved in 0.1 mL ofwater or 50 mM potassium phosphate buffer (pH=5, 6 or 7.4). Tc-99mgluceptate was prepared by reconstituting a Glucoscan vial (E.I. DuPontde Nemours, Inc.) with 1.0 mL of Tc-99m sodium pertechnetate containingup to 200 mCi and allowed to stand for 15 minutes at room temperature.25 μl of Tc-99m gluceptate was then added to the peptide and thereaction allowed to proceed at room temperature or at 100° C. for 15-30min and then filtered through a 0.2 μm filter.

The Tc-99m labeled peptide purity was determined by HPLC using theconditions described in the Footnotes in Table I. Radioactive componentswere detected by an in-line radiometric detector linked to anintegrating recorder. Tc-99m gluceptate and Tc-99m sodium pertechnetateelute between 1 and 4 minutes under these conditions, whereas the Tc-99mlabeled peptide eluted after a much greater amount of time.

The following Table illustrates successful Tc-99m labeling of peptidesprepared according to Example 2 using the method described herein.

    __________________________________________________________________________                            FABMS                                                                             Radiochemical                                                                        HPLC                                       Pentides                MH.sup.+                                                                          Yield(%)*                                                                            R.sub.τ (min)**                        __________________________________________________________________________    formyl-MLFC.sub.Acm G.Pica                                                                             760                                                                              100.sup.2                                                                            10.9, 11.5, 12.2.sup.1                     Pic.GC.sub.Acm (VGVAPG).sub.3 amide                                                                   1795                                                                              100.sup.2                                                                            12.4.sup.3                                 Pic.GC.sub.Acm (VPGVG).sub.4 amide                                                                    1992                                                                              100.sup.2                                                                            12.0.sup.1                                 Pic.GC.sub.Acm RALVDTLKFVTQAEGAKamide                                                                 2183                                                                               95.sup.2                                                                            17.2.sup.3                                 Pic.GC.sub.Acm RALVDTEFKVKQEAGAKamide                                                                 2226                                                                               96.sup.2                                                                            15.5.sup.3                                 Pic.GC.sub.Acm PLARITLPDFRLPEIAIPamide                                                                2368                                                                               92.sup.2                                                                            19.2.sup.1                                 Pic.GC.sub.Acm GQQHHLGGAKAGDV                                                                         1838                                                                               48.sup.2                                                                            12.8-16.6.sup.2                            Pic.GC.sub.Acm PLYKKIIKKLLES                                                                          1910                                                                               81.sup.2                                                                            10.7-14.9.sup.2                            Pic.GC.sub.Acm LRALVDTLKamide                                                                         1363                                                                               92.sup.6                                                                            13.0-14.9.sup.2                            Pic.GC.sub.Acm GGGLRALVDTLKamide                                                                      1535                                                                              100.sup.1                                                                            15.6.sup.2                                 Pic.GC.sub.Acm GGGLRALVDTLKFVTQAEGAKamide                                                             2354                                                                               92.sup.5                                                                            15.1.sup.2                                 Pic.GC.sub.Acm GGGRALVDTLKALVDTLamide                                                                 2035                                                                               86.sup.6                                                                            14.5.sup.2                                 Pic.GC.sub.Acm GHRPLDKKREEAPSLRPAPPPISGGGYR                                                           3377                                                                               94.sup.6                                                                            11.3.sup.2                                 Pic.GC.sub.Acm PSPSPIHPAHHKRDRRQamide                                                                 2351                                                                               94.sup.6                                                                            11.2, 14.4.sup.2                           Pic.GC.sub.Acm GGGF.sub.D.Cpa.YW.sub.D KTFTamide                                                      1681                                                                               98.sup.3                                                                            13.8-16.8.sup.2                             ##STR6##               1217                                                                               69.sup.2                                                                            6.6-13.7.sup.2                              Pic.SC.sub.Acm SYNRGDSTC(S-maleimido)CH.sub.2 CH.sub.2 !.sub.3 N.sup.a                               4488                                                                               99.sup.2                                                                            10.4, 11.2.sup.2                           Pic.GC.sub.Mob GGGLRALVDTLKamide                                                                      1471                                                                              100.sup.6                                                                            11.9.sup.4                                 Pic.GCGGGLRALVDTLKamide 1350                                                                              100.sup.6                                                                            11.2, 11.6.sup.4                           Pic.GCYRALVDThKFVTQAEGAKamide                                                                         2275                                                                               95.sup.3                                                                            18.6, 19.1.sup.2                           Pic.GC(VGVAPG).sub.3 amide                                                                            1724                                                                               95.sup.3                                                                            17.3.sup.2                                 BAT-RALVDTLKFVTQAEGAKamide                                                                            2165                                                                               98.sup.3                                                                            19.0.sup.2                                 __________________________________________________________________________     *Superscripts refer to the following labeling conditions:                     .sup.1 The peptide is dissolved in 50 mM potassium phosphate buffer (pH       7.4) and labeled at room temperature.                                         .sup.2 The peptide is dissolved in 50 mM potassium phosphate buffer (pH       7.4) and labeled at 100° C.                                            .sup.3 The peptide is dissolved in water and labeled at room temperature.     .sup.4 The peptide is dissolved in water and labeled at 100° C.        .sup.5 The peptide is dissolved in 50 mM polassium phosphate buffer (pH       6.0) and labeled at 100° C.                                            .sup.6 The peptide is dissolved in 50 mM potassium phosphate buffer (pH       5.0) and labeled at room temperature.                                         **HPLC methods (indicated by superscript after R.sub.τ):                  general: solvent A = 0.1% CF.sub.3 COOH/H.sub.2 O                             solvent B.sub.70 = 0.1% CF.sub.3 COOH/70% CH.sub.3 CN/H.sub.2 O               solvent B.sub.90 = 0.1% CF.sub.3 COOH/90% CH.sub.3 CN/H.sub.2 O               solvent flow rate = 1mL/min                                              

Vydak column=Vydak 218TP54 RP-18, 5μ×220 mm×4.6mm analytical column withguard column Brownlee column=Brownlee Spheri-5 RP-18, 5μ×220 mm×4.6mmcolumn

    ______________________________________                                        Method 1:                                                                             Brownlee column                                                                            100% A to 100% B.sub.70 in 10 min                        Method 2:                                                                             Vydak column 100% A to 100% B.sub.90 in 10 min                        Method 3:                                                                             Vydak column 100% A to 100% B.sub.70 in 10 min                        Method 4:                                                                             Brownlee column                                                                            100% A to 100% B.sub.90 in 10 min                        ______________________________________                                    

Single-letter abbreviations for amino acids can be found in G. Zubay,Biochemistry (2d. ed.), 1988 (MacMillen Publishing: New York) p.33;Ac=acetyl; Pic=picolinoyl (pyridine-2-carbonyl); Acm=acetamidomethyl;Mob=4-methoxybenzyl; Pica=picolylamine (2-aminomethyl)pyridine);Apc=L-S-3-aminopropyl)cysteine; F_(D) =D-phenylalanine; Y_(D)=D-tyrosine; Cpa=L-4-chloropheayl)alanine; BAT=HSC(CH₃)₂ CH₂ NHCH₂ CH₂N(CH₂ CH₂ CH₂ CH₂ CO--)CH₂ C(CH₃)₂ SH

The structure of this compound is as follows: ##STR7##

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 19                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 23..24                                                          (D) OTHER INFORMATION: /label=AMIDE                                           /note= "The carboxyl terminus is an amide."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ValGlyValAlaProGlyValGlyValAlaProGlyValGlyValAla                              151015                                                                        ProGly                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 19..20                                                          (D) OTHER INFORMATION: /label=AMIDE                                           /note= "The carboxyl terminus is an amide."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ValProGlyValGlyValProGlyValGlyValProGlyValGlyVal                              151015                                                                        ProGlyValGly                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 16..17                                                          (D) OTHER INFORMATION: /label=AMIDE                                           /note= "The carboxyl terminus is an amide."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ArgAlaLeuValAspThrLeuLysPheValThrGlnAlaGluGlyAla                              151015                                                                        Lys                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 16..17                                                          (D) OTHER INFORMATION: /label=AMIDE                                           /note= "The carboxyl terminus is an amide."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ArgAlaLeuValAspThrGluPheLysValLysGlnGluAlaGlyAla                              151015                                                                        Lys                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 17..18                                                          (D) OTHER INFORMATION: /label=AMIDE                                           /note= "The carboxyl terminus is an amide."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ProLeuAlaArgIleThrLeuProAspPheArgLeuProGluIleAla                              151015                                                                        IlePro                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GlyGlnGlnHisHisLeuGlyGlyAlaLysAlaGlyAspVal                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ProLeuTyrLysLysIleIleLysLysLeuLeuGluSer                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8..9                                                            (D) OTHER INFORMATION: /label=AMIDE                                           /note= "The carboxyl terminus is an amide."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LeuArgAlaLeuValAspThrLeuLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11..12                                                          (D) OTHER INFORMATION: /label=AMIDE                                           /note= "The carboxyl terminus is an amide."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlyGlyGlyLeuArgAlaLeuValAspThrLeuLys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 20..21                                                          (D) OTHER INFORMATION: /label=AMIDE                                           /note= "The carboxyl terminus is an amide."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GlyGlyGlyLeuArgAlaLeuValAspThrLeuLysPheValThrGln                              151015                                                                        AlaGluGlyAlaLys                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 16..17                                                          (D) OTHER INFORMATION: /label=AMIDE                                           /note= "The carboxyl terminus is an amide."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GlyGlyGlyArgAlaLeuValAspThrLeuLysAlaLeuValAspThr                              151015                                                                        Leu                                                                           (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GlyHisArgProLeuAspLysLysArgGluGluAlaProSerLeuArg                              151015                                                                        ProAlaProProProIleSerGlyGlyGlyTyrArg                                          2025                                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 16..17                                                          (D) OTHER INFORMATION: /label=AMIDE                                           /note= "The carboxyl terminus is an amide."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ProSerProSerProIleHisProAlaHisHisLysArgAspArgArg                              151015                                                                        Gln                                                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4..7                                                            (D) OTHER INFORMATION: /label=Variantaas                                      /note= "The phenylalanine and tryptophan residues                             are the D stereoisomers, residue X is                                         L-(4- chlorophenyl) alanine, and the carboxyl termimus..."                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GlyGlyGlyPheXaaTyrTrpLysThrPheThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 3..9                                                            (D) OTHER INFORMATION: /label=Variant                                         /note= "Residue X is L- S-(3-aminopropyl)                                     cysteine"                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GlyGlyCysAsnProXaaGlyAspCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11..12                                                          (D) OTHER INFORMATION: /label=AMIDE                                           /note= "The carboxyl terminus is an amide."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GlyGlyGlyLeuArgAlaLeuValAspThrLeuLys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11..12                                                          (D) OTHER INFORMATION: /label=AMIDE                                           /note= "The carboxyl terminus is an amide."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GlyCysGlyGlyGlyLeuArgAlaLeuValAspThrLeuLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 19..20                                                          (D) OTHER INFORMATION: /label=AMIDE                                           /note= "The carboxyl terminus is an amide."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GlyCysTyrArgAlaLeuValAspThrLeuLysPheValThrGlnAla                              151015                                                                        GluGlyAlaLys                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 19..20                                                          (D) OTHER INFORMATION: /label=AMIDE                                           /note= "The carboxyl terminus is an amide."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GlyCysValGlyValAlaProGlyValGlyValAlaProGlyValGly                              151015                                                                        ValAlaProGly                                                                  20                                                                            __________________________________________________________________________

What is claimed is:
 1. A composition comprising a specific bindingpeptide and a radiolabel-binding moiety covalently linked thereto,wherein the moiety forms a complex with technetium-99m, and wherein thecomplex is electrically neutral, thereby avoiding interference of thecomplex with specific binding of the peptide to a target in vivo.
 2. Thecomposition of claim 1 wherein the peptide and the radiolabel-bindingmoiety are covalently linked through one or more amino acids.
 3. Thecomposition of claim 1 wherein the peptide is selected from the groupconsisting of:formyl-MLF, (VGVAPG)₃ amide, (VPGVG)₄ amide,RALVDTLKFVTQAEGAKamide, RALVDTEFKVKQEAGAKamide, PLARITLPDFRLPEIAIPamide,GQQHHLGGAKAGDV, PLYKKIIKKLLES, LRALVDTLKamide, GGGLRALVDTLKamide,GGGLRALVDTLKFVTQAEGAKamide, GGGRALVDTLKALVDTLamide,GHRPLDKKREEAPSLRPAPPPISGGGYR, PSPSPIHPAHHKRDRRQamide,GGGF_(D).Dpa.YW_(D) KTFTamide, ##STR8## (SYNRGDSTC(S-maleimido)CH₂ CH₂-)₃ N, GGGLRALVDTLKamide, GCGGGLRALVDTLKamide,GCYRALVDTLKFVTQAEGAKamideand GC(VGVAPG)₃ amide.
 4. The composition ofclaim 1, wherein the complex is formed with technetium-99m in thepresence of a reducing agent.
 5. The composition of claim 4, wherein thereducing agent is selected from the group consisting of a dithioniteion, a stannous ion and a ferrous ion.
 6. A composition comprising aspecific binding peptide covalently linked to a radiolabel-bindingmoiety selected from the group consisting of: ##STR9## wherein X=H or aprotecting group;(amino acid)=any amino acid;and a bisamino bisthiolradiolabel-binding moiety having a formula selected from the groupconsisting of: ##STR10## wherein each R is independently H, CH₃ or C₂ H₅; each (pgp)^(S) is independently a thiol protecting group or H; m, nand p are independently 2 or 3; A=linear or cyclic lower alkyl, aryl,heterocyclyl, combinations or substituted derivatives thereof;and##STR11## wherein each R is independently H, CH₃ or C₂ H₅ ; m, n and pare independently 2 or 3; A=linear or cyclic lower alkyl, aryl,heterocyclyl, combinations or substituted derivatives thereof; V=H or--CO-peptide; R'=H or peptide;and wherein when V=H, R'=peptide and whenR'=H, V=--CO-peptide; wherein the radiolabel-binding moiety forms acomplex with technetium-99m and the complex is electrically neutral,thereby avoiding interference of the complex with specific binding ofthe peptide to a target in vivo.
 7. The composition of claim 6 whereinthe complex is formed by ligand exchange of a prereduced technetium-99mcomplex.
 8. The composition of claim 6 wherein the peptide and theradiolabel-binding moiety are covalently linked through one or moreamino acids.
 9. The composition of claim 6 wherein the peptide isselected from the group consisting of:formyl-MLF, (VGVAPG)₃ amide,(VPGVG)₄ amide, RALVDTLKFVTQAEGAKamide, RALVDTEFKVKQEAGAKamide,PLARITLPDFRLPEIAIPamide, GQQHHLGGAKAGDV, PLYKKIIKKLLES, LRALVDTLKamide,GGGLRALVDTLKamide, GGGLRALVDTLKFVTQAEGAKamide, GGGRALVDTLKALVDTLamide,GHRPLDKKREEAPSLRPAPPPISGGGYR, PSPSPIHPAHHKRDRRQamide,GGGF_(D).Cpa.YW_(D) KTFTamide, ##STR12## (SYNRGDSTC(S-maleimido)CH₂ CH₂--)₃ N, GGGLRALVDTLKamide, GCGGGLRALVDTLKamide,GCYRALVDTLKFVTQAEGAKamideand GC(VGVAPG)₃ amide.
 10. The composition ofclaim 6 wherein the amino acid is glycine and wherein X is anacetamidomethyl protecting group in the radiolabel-binding moietieshaving formula: ##STR13##
 11. The composition of claim 6, wherein thecomplex is formed with technetium-99m in the presence of a reducingagent.
 12. The composition of claim 11, wherein the reducing agent isselected from the group consisting of a dithionite ion, a stannous ionand a ferrous ion.